S. Diederichs (Heidelberg, DE)
Orthopaedic University Hospital Heidelberg Research Centre for Experimental OrthopaedicsPresenter Of 2 Presentations
16.1.3 - Enhanced Cartilage Tissue Yield from Induced Pluripotent Stem Cells by Initial WNT/beta-Catenin Activation
Abstract
Purpose
Induced pluripotent stem cells (iPSCs) are promising for cartilage tissue engineering as they are unlimited in supply. We have established chondrocyte differentiation of iPSCs, but high cell loss currently compromises tissue yield. During embryo development, cell survival and proliferation are regulated by several pathways including WNT/β-catenin. WNT/β-catenin activation initiates mesoderm commitment and hence might be important for chondrocyte differentiation. We here asked, whether short stimulation of WNT/β-catenin signalling at initiation of differentiation would improve cell survival and tissue yield during subsequent iPSC chondrogenesis.
Methods and Materials
Two human iPSC-lines (IMR90,CB) were differentiated towards mesoderm (14days) using bFGF/serum on matrigel/gelatin followed by 42days of chondrogenic 3D-pellet culture with 10ng/ml TGF-β1. One group was stimulated with a WNT/β-catenin pulse (5µM CHIR99021) for 24h at the start of mesoderm differentiation (d0). Effects were analyzed by Western blot, qPCR, cDNA-microarray, histology, aggregation assay and DNA quantification.
Results
Initial CHIR treatment significantly increased the number of PDGFRα-positive cells at d7 compared to controls. Accordingly, mRNA-levels of mesoderm markers were significantly elevated at d14 and common ectodermal markers reduced, demonstrating an enhanced mesoderm commitment of CHIR-treated iPSCs. While controls quickly formed multiple free-floating small aggregates and only few cells attached to the plastic surface, CHIR-treated cells aggregated into a plastic-adherent cell sheet, which subsequently condensed into one large pellet over 2-14days. In line, CHIR-treated cells expressed ECM and adhesion-related genes at higher levels than controls at d14. As a consequence of improved pellet formation, DNA amounts in the CHIR-group remained significantly higher than in controls during chondrogenic culture, resulting in significantly larger pellets.
Conclusion
Initial WNT activation improved mesoderm commitment; and we demonstrate for the first time that, acting via stimulated cell proliferation, ECM-expression and cell-aggregation, WNT-pulsing is key to rescue low tissue yield during chondrogenesis. This advancement can be highly beneficial for clinical cartilage regeneration, disease modelling and drug screening.
16.1.4 - Differential PI3K/AKT Activity in Endochondral vs. Chondral Development In Vitro
Abstract
Purpose
A main limitation of cartilage engineering with multipotent stromal cells (MSC) is their inherent endochondral development. Current treatments like WNT inhibition can only reduce chondrocyte hypertrophy, but not induce an articular chondrocyte (AC)-like phenotype in MSC. PI3K/AKT signaling is essential for cartilage neogenesis and chondrocyte hypertrophy in the growth plate. Yet, its role for hypertrophic differentiation of MSC in vitro remains unclear. Aim was to uncover if different PI3K/AKT activity in MSC-derived hypertrophic chondrocytes vs. non-hypertrophic AC might indicate PI3K/AKT inhibition as promising to reduce hypertrophy during MSC chondrogenesis.
Methods and Materials
Human expanded MSC and AC were subjected to chondrogenic 3D culture for 42 days and AKT activity was detected via phospho-AKT Western blot. PI3K/AKT signaling was inhibited by LY294002 (0.25µM - 25µM) from day 21 on. Differentiation was assessed via histology, qPCR, proteoglycan quantification and alkaline phosphatase activity. To evaluate AKT activity under anti-hypertrophic stimulation, MSC pellets were co-treated with the WNT inhibitor IWP-2 (5µM, d14-d42).
Results
Unlike AC, MSC upregulated AKT activity during chondrogenesis in parallel to hypertrophy (figure 1), reaching significantly higher levels than AC from d21 on. LY dose-dependently reduced hypertrophic (IHH, PTHR1, COL10A1 mRNA) along with chondrogenic markers (COL2A1, ACAN) and proteoglycan deposition. In line, LY reduced TGFβ-induced pSMAD2/3 and SOX9 protein. Importantly, no LY dose was capable to selectively target hypertrophic but not chondrocyte markers. This indicated that PI3K/AKT activity played primarily a pro-chondrogenic role and may not regulate hypertrophy. In line with this observation, pAKT protein levels became also upregulated in MSC under anti-hypertrophic treatment with IWP-2.
Conclusion
Although the increasing AKT activity in MSC but not in AC suggested its relevance for endochondral differentiation, we here demonstrated that MSC chondrogenesis strongly depends on PI3K/AKT activation. This indicates that induction and maintenance of PI3K/AKT activity will be crucial for future therapeutic success of MSC-based cartilage regeneration.